Dehalogenases, Nucleic Acids Encoding Them and Methods for Making and Using Them

ABSTRACT

The invention relates to haloalkane dehalogenases and to polynucleotides encoding the haloalkane dehalogenases. In addition methods of designing new dehalogenases and method of use thereof are also provided. The dehalogenases have increased activity and stability at increased pH and temperature.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. (U.S. Ser. No.) 12/683,906, filed Jan. 7, 2010, now pending; whichis a divisional application of U.S. Ser. No. 11/418,828, filed May 5,2006, now U.S. Pat. No. 7,671,189; which is a divisional application ofU.S. Ser. No. 10/000,997, filed Nov. 30, 2001, now U.S. Pat. No.7,078,504; which claims benefit of priority under 35 U.S.C. §119(e) toProvisional patent application U.S. Ser. No. 60/250,897, filed Dec. 1,2000. Each of the aforementioned applications is explicitly incorporatedherein by reference in its entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §1730 II.B.2(a)(C), is incorporated herein by reference inits entirety for all purposes. The sequence listing is identified on theelectronically filed text file as follows:

File Name Date of Creation Size (bytes) D15501D3_SequenceListing.txtApr. 9, 2012 298,496 bytes

FIELD OF THE INVENTION

This invention relates generally to enzymes, polynucleotides encodingthe enzymes, the use of such polynucleotides and polypeptides, and morespecifically to enzymes having haloalkane dehalogenase activity.

BACKGROUND ART

Environmental pollutants consist of a large quantity and variety ofchemicals; many of these are toxic, environmental hazards that weredesignated in 1979 as priority pollutants by the U.S. EnvironmentalProtection Agency. Microbial and enzymatic biodegradation is one methodfor the elimination of these pollutants. Accordingly, methods have beendesigned to treat commercial wastes and to bioremediate pollutedenvironments via microbial and related enzymatic processes.

Unfortunately, many chemical pollutants are either resistant tomicrobial degradation or are toxic to potential microbial-degraders whenpresent in high concentrations and certain combinations.

Haloalkane dehalogenase belongs to the alpha/beta hydrolase fold familyin which all of the enzymes share similar topology, reaction mechanisms,and catalytic triad residues (Krooshof, et al., Biochemistry36(31):9571-9580, 1997). The enzyme cleaves carbon-halogen bonds inhaloalkanes and halocarboxylic acids by hydrolysis, thus converting themto their corresponding alcohols. This reaction is important fordetoxification involving haloalkanes such as ethylchloride,methylchloride, and 1,2-dichloroethane, which are considered prioritypollutants by the Environmental Protection Agency (Rozeboom, H., Kingma,J., Janssen, D., Dijkstra, B., Crystallization of HaloalkaneDehalogenase from Xanthobacter autotrophicus GJ10, J Mol Biol, 200 (3),611-612 (1988)).

The haloalkane dehalogenases are produced by microorganisms that cangrow entirely on chlorinated aliphatic compounds. No metal or oxygen isneeded for activity: water is the sole substrate.

Xanthobacter autotrophicus GJ10 is a nitrogen-fixing bacteria thatutilizes 1,2-dichloroethane and a few other haloalkane andhalocarboxylic acids for growth (Rozeboom, et al., J Mol Biol, 2003:611-612, 1988; Keuning, et al., J Bacteriol, 163(2):635-639, 1985). Itis the most well-studied dehalogenase because it has a known catalyticreaction mechanism, activity mechanism and crystal-structure (Schanstra,et al., J Biol Chem, 271(25):14747-14753, 1996).

The organism produces two different dehalogenases. One dehalogenase isfor halogenated alkanes and the other for halogenated carboxylic acids.Most harmful halogenated compounds are industrially produced for use ascleaning agents, pesticides, and solvents. The natural substrate ofXanthobacter autotrophicus is 1,2-dichloroethane. This haloalkane isoften used in vinyl production.

Enzymes are highly selective catalysts. Their hallmark is the ability tocatalyze reactions with exquisite stereo-, regio-, andchemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, enzymes are remarkably versatile. They can betailored to function in organic solvents, operate at extreme pH's andtemperatures, and catalyze reactions with compounds that arestructurally unrelated to their natural, physiological substrates.

Enzymes are reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Moreover, unlike traditional chemical catalysts, enzymes arehighly enantio- and regio-selective. The high degree of functional groupspecificity exhibited by enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes are also capable of catalyzing many diverse reactions unrelatedto their physiological function in nature. For example, peroxidasescatalyze the oxidation of phenols by hydrogen peroxide. Peroxidases canalso catalyze hydroxylation reactions that are not related to the nativefunction of the enzyme. Other examples are proteases which catalyze thebreakdown of polypeptides. In organic solution some proteases can alsoacylate sugars, a function unrelated to the native function of theseenzymes.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds.

Each biocatalyst is specific for one functional group, or severalrelated functional groups, and can react with many starting compoundscontaining this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original compoundcan be produced with each iteration of biocatalytic derivitization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods. (For further teachings on modification ofmolecules, including small molecules, See PCT/US94/09174, hereinincorporated by reference in its entirety).

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

The invention provides an isolated nucleic acid having a sequence as setforth in SEQ ID NO's: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 43, 45, 47 and variants thereof having at least 50%sequence identity to SEQ ID NO.: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 43, 45 or 47 and encoding polypeptideshaving dehalogenase activity.

One aspect of the invention is an isolated nucleic acid having asequence as set forth in SEQ ID NO's: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 43, 45, 47 (hereinafter referred toas “Group A nucleic acid sequences”), sequences substantially identicalthereto, and sequences complementary thereto.

Another aspect of the invention is an isolated nucleic acid including atleast 10 consecutive bases of a sequence as set forth in Group A nucleicacid sequences, sequences substantially identical thereto, and thesequences complementary thereto.

In yet another aspect, the invention provides an isolated nucleic acidencoding a polypeptide having a sequence as set forth in SEQ ID NO's: 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 44,46, 48 and variants thereof encoding a polypeptide having dehalogenaseactivity and having at least 50% sequence identity to such sequences.

Another aspect of the invention is an isolated nucleic acid encoding apolypeptide or a functional fragment thereof having a sequence as setforth in SEQ ID NO's: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 44, 46, 48 (hereinafter referred to as “Group Bamino acid sequences”), and sequences substantially identical thereto.

Another aspect of the invention is an isolated nucleic acid encoding apolypeptide having at least 10 consecutive amino acids of a sequence asset forth in Group B amino acid sequences, and sequences substantiallyidentical thereto.

In yet another aspect, the invention provides a purified polypeptidehaving a sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto.

Another aspect of the invention is an isolated or purified antibody thatspecifically binds to a polypeptide having a sequence as set forth inGroup B amino acid sequences, and sequences substantially identicalthereto.

Another aspect of the invention is an isolated or purified antibody orbinding fragment thereof, which specifically binds to a polypeptidehaving at least 10 consecutive amino acids of one of the polypeptides ofGroup B amino acid sequences, and sequences substantially identicalthereto.

Another aspect of the invention is a method of making a polypeptidehaving a sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto. The method includesintroducing a nucleic acid encoding the polypeptide into a host cell,wherein the nucleic acid is operably linked to a promoter, and culturingthe host cell under conditions that allow expression of the nucleicacid.

Another aspect of the invention is a method of making a polypeptidehaving at least 10 amino acids of a sequence as set forth in Group Bamino acid sequences, and sequences substantially identical thereto. Themethod includes introducing a nucleic acid encoding the polypeptide intoa host cell, wherein the nucleic acid is operably linked to a promoter,and culturing the host cell under conditions that allow expression ofthe nucleic acid, thereby producing the polypeptide.

Another aspect of the invention is a method of generating a variantincluding obtaining a nucleic acid having a sequence as set forth inGroup A nucleic acid sequences, sequences substantially identicalthereto, sequences complementary to the sequences of Group A nucleicacid sequences, fragments comprising at least 30 consecutive nucleotidesof the foregoing sequences, and changing one or more nucleotides in thesequence to another nucleotide, deleting one or more nucleotides in thesequence, or adding one or more nucleotides to the sequence.

Another aspect of the invention is a computer readable medium havingstored thereon a sequence as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto.

Another aspect of the invention is a computer system including aprocessor and a data storage device wherein the data storage device hasstored thereon a sequence as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto, or apolypeptide having a sequence as set forth in Group B amino acidsequences, and sequences substantially identical thereto.

Another aspect of the invention is a method for comparing a firstsequence to a reference sequence wherein the first sequence is a nucleicacid having a sequence as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide code ofGroup B amino acid sequences, and sequences substantially identicalthereto. The method includes reading the first sequence and thereference sequence through use of a computer program which comparessequences; and determining differences between the first sequence andthe reference sequence with the computer program.

Another aspect of the invention is a method for identifying a feature ina sequence as set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto, or a polypeptide having a sequence asset forth in Group B amino acid sequences, and sequences substantiallyidentical thereto, including reading the sequence through the use of acomputer program which identifies features in sequences; and identifyingfeatures in the sequence with the computer program.

Another aspect of the invention is an assay for identifying fragments orvariants of Group B amino acid sequences, and sequences substantiallyidentical thereto, which retain the enzymatic function of thepolypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto. The assay includes contacting thepolypeptide of Group B amino acid sequences, sequences substantiallyidentical thereto, or polypeptide fragment or variant with a substratemolecule under conditions which allow the polypeptide fragment orvariant to function, and detecting either a decrease in the level ofsubstrate or an increase in the level of the specific reaction productof the reaction between the polypeptide and substrate therebyidentifying a fragment or variant of such sequences.

In yet another aspect, the invention provides a method for synthesizingglycerol. The method includes contacting trichloropropane ordichloropropanol with a polypeptide having at least 70% homology to asequence selected from the group consisting of Group B amino acidsequences and sequences substantially identical thereto, and havingdehalogenase activity, under conditions to synthesize glycerol.

In yet another aspect, the invention provides a method for producing anoptically active halolactic acid. The method includes contacting adihalopropionic acid with a polypeptide having at least 70% homology toa sequence selected from the group consisting of Group B amino acidsequences and sequences substantially identical thereto, and havingdehalogenase activity, under conditions to produce optically activehalolactic acid.

In yet another aspect, the invention provides a method forbioremediation by contacting an environmental sample with a polypeptidehaving at least 70% homology to a sequence selected from the groupconsisting of Group B amino acid sequences and sequences substantiallyidentical thereto, and having dehalogenase activity.

In another aspect, the invention provides a method for removing ahalogenated contaminant or halogenated impurity from a sample. Themethod includes contacting the sample with a polypeptide having at least70% homology to a sequence selected from the group consisting of Group Bamino acid sequences and sequences substantially identical thereto, andhaving dehalogenase activity.

In yet another aspect, the invention provides a method for synthesizinga diol, by contacting a dihalopropane or monohalopropanol with apolypeptide having at least 70% homology to a sequence selected from thegroup consisting of Group B amino acid sequences and sequencessubstantially identical thereto, and having dehalogenase activity, underconditions to synthesize the diol.

In yet another aspect, the invention provides a method fordehalogenating a halo-substituted cyclic hydrocarbyl. The methodincludes contacting the halo-substituted cyclic hydrocarbyl with apolypeptide having at least 70% homology to a sequence selected from thegroup consisting of Group B amino acid sequences and sequencessubstantially identical thereto, and having dehalogenase activity, underconditions to dehalogenate the halo-substituted cyclic hydrocarbyl.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one embodiment of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one embodiment of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 shows an alignment of the polypeptide sequences of the invention.A=SEQ ID NO:4; B=SEQ ID NO:2; C═SEQ ID NO:6; rhod2=SEQ ID NO:40;myco4=SEQ ID NO:42; Consensus=SEQ ID NO:49.

FIGS. 6A-6R-2 shows sequences of the invention (SEQ ID NO's: 9-38 and43-48).

FIG. 7 shows an example of the formation of glycerol using thedehalogenases of the invention as well as the formation of1,2-propanediol or 1,3-propanediol using the dehalogenases of theinvention.

FIG. 8 shows an example of the dehalogenation of a halo-substitutedcyclic hydrocarbyl using the dehalogenases of the invention.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to haloalkane dehalogenase polypeptides andpolynucleotides encoding them as well as methods of use of thepolynucleotides and polypeptides. As used herein, the terminology“haloalkane dehalogenase” encompasses enzymes having hydrolase activity,for example, enzymes capable of catalyzing the hydrolysis of haloalkanesvia an alkyl-enzyme intermediate.

The polynucleotides of the invention have been identified as encodingpolypeptides having dehalogenase activity and in particular embodimentshaloalkane dehalogenase activity.

The dehalogenases and polynucleotides encoding the dehalogenases of theinvention are useful in a number of processes, methods, andcompositions. For example, as discussed above, a dehalogenase can beused to remedy an environment contaminated with aliphaticorganochlorine, degrade the herbicide dalapon, degrade halogenatedorganic acids as well as soil and water remediation, and treat bydegradation halogenated organic acid in the soil and water. Furthermore,a dehalogenase of the invention can be used to remove impurities inindustrial processes, in the environment, and in medicaments. Forexample, a dehalogenase can be used to decompose haloalkanoic acidimpurities in various samples including, for example, surfactants,carboxymethyl cellulose or thioglycolic acid salts. In yet anotheraspect, the dehalogenases of the invention can be used in the formationof medicines, agrochemical and ferroelectric liquids by allowingoxidative dehalogenation of specific 1,2-diol or racemichalogenohydrins. For example, a dehalogenase can be used in thesynthesis of optically active glycidic and lactic acids (e.g., betahalolactic acid) by treating an 1,θ-dihalopropionic acid (e.g.,dichloropropionic acid) with a dehalogenase. The dehalogenases of theinvention can also be used in the production of active(S)-(+)-3-halo-1,2-propanediol or (R)-(−)-3 halo-1,2 propanediol from1,3-dihalo-2-propanol. (S)-(+)-3 halo-1,2-propanediol is useful as a rawmaterial for physiological and medical treatments and medicaments. Forexample, a dehalogenase of the invention can be contactedtrichloropropanediol (TCP) or dichloropropanediol (DCP) under conditionsand for a time sufficient to allow oxidative dehalogenation to form, forexample, glycerol (e.g., DCP or TCP to glycerol) (see, for example, FIG.7). Various diols can be produced using the methods of the invention andthe enzymes of the invention. In addition, the methods and compositionsof the invention can be applied to halogenated aromatic compounds. Forexample, the compositions of the invention can be used to dehalogenate ahalo-substituted cyclic hydrocarbyl as depicted in FIG. 8. Examples ofcyclic hydrocarbyl compounds include cycloalkyl, cycloalkenyl,cycloalkadienyl, cycloalkatrienyl, cycloalkynyl, cycloalkadiynyl,aromatic compounds, spiro hydrocarbons wherein two rings are joined by asingle atom which is the only common member of the two rings (e.g.,spiro[3,4]octanyl, and the like), bicyclic hydrocarbons wherein tworings are joined and have at least two atoms in common (e.g.,bicyclo[3.2.1]octane, bicyclo[2.2.1]hept-2-ene, and the like), ringassemblies wherein two or more cyclic systems (i.e., single rings orfused systems) are directly joined to each other by single or doublebonds, and the number of such ring junctions is one less than the numberof cyclic systems involved (e.g., biphenylyl, biphenylylene, radicals orp-terphenyl, cyclohexylbenzyl, and the like), polycyclics, and the like.

Haloalkane Dehalogenase

Overall Structure

Haloalkane dehalogenase from Xanthobacter autotrophicus is composed of310 amino acids and consists of a single polypeptide chain with amolecular weight of 36,000. The monomeric enzyme is spherical andcomposed of two domains. The main domain has an alpha/beta hydrolasefold structure with a mixed beta sheet of 8 strands order 12435678;strand 2 is antiparallel to the rest. The second domain is analpha-helical cap which lies on top of the main domain. (Keuning, etal., J Bacteriol 163(2):635-639, 1985) As described in further detailherein, mutagenesis have done to modify the activity of the enzyme, forexample, by mutating specific residues of the cap domain (Krooshof, etal., Biochemistry 36(31):9571-9580, 1997).

The active site of the enzyme in Xanthobacter autotrophicus, consistingof three catalytic residues (Asp 124, His 289, and Asp 260), is foundbetween the two domains in an internal hydrophobic cavity. NucleophilicAsp 124 and the general base His 289, located after beta-strands 5 and 8respectively, are fully conserved in the alpha/beta hydrolase family,while Asp 260 is not. The active site is lined with 10 hydrophobicresidues: 4 phenylalanines; 2 tryptophans; 2 leucines; 1 valine; and 1proline. (Schanstra, et al., J Biol Chem 271(25):14747-14753, 1996).

During enzymatic hydrolysis of a substrate, haloalkane dehalogenaseforms a covalent intermediate formed by nucleophilic substitution withAsp 124 that is hydrolyzed by a water molecule that is activated by His289. (Verschueren, et al., Nature 363(6431):693-698, 1993). The role ofAsp 260, which is the third member of a catalytic triad common todehalogenase enzymes, has been studied by site-directed mutagenesis.Mutation of Asp 260 to asparagine resulted in a catalytically inactiveD260N mutant, which demonstrates that the triad acid Asp 260 isessential for dehalogenase activity in the wild-type enzyme.Furthermore, Asp 260 has an important structural role, since the D260Nenzyme accumulated mainly in inclusion bodies during expression, andneither substrate nor product could bind in the active-site cavity.Activity for brominated substrates was restored to D260N by replacingAsn 148 with an aspartic or glutamic acid. Both double mutantsD260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-foldhigher Km values for 1,2-dibromoethane compared to the wild-type enzyme.Pre-steady-state kinetic analysis of the D260N+N148E double mutantshowed that the decrease in kcat was mainly caused by a 220-foldreduction of the rate of carbon-bromine bond cleavage and a 10-folddecrease in the rate of hydrolysis of the alkyl-enzyme intermediate. Onthe other hand, bromide was released 12-fold faster and via a differentpathway than in the wild-type enzyme. Molecular modeling of the mutantshowed that Glu 148 indeed could take over the interaction with His 289and that there was a change in charge distribution in the tunnel regionthat connects the active site with the solvent. (Krooshof, et al.,Biochemistry 36(31):9571-9580, 1997).

The first step in degradation of the harmful halogenated compoundsutilizes haloalkane dehalogenase. The dehalogenase catalysis occurs as atwo step-mechanism involving an ester intermediate. No energy isrequired for hydrolytic dehalogenases; therefore, it is a simple way todetoxify organic matter since the halogen, which causes the toxicity, islost. A catalytic triad (Asp-His-Asp), along with an aspartatecarboxylate (Asp 124), are the focal point of the reaction. Thesubstrate binds to the active site cavity and the Cl-alpha complexreacts with the side chain NH groups of Trp 172 and Trp 175. As a firststep a halogen from the substrate is displaced by the nucleophilicaspartate, resulting in an intermediate covalent ester. His 289 thenactivates a water molecule which hydrolyzes the ester. As a result, analcohol and halide are displaced from the active site. The two stepmechanism involving nucleophilic Asp 124 and water hydrolysis of theester intermediate is consistent with other alpha/beta hydrolase foldenzymes.

Haloalkane dehalogenase breaks carbon-halogen bonds in aliphaticcompounds. Results show that the enzyme reaction with C—Cl bond isslower than that of other C-halide bonds, such as C—Br bonds. Theability of the leaving group is the explanation for the difference. Therate limiting step for 1,2-dichloroethane and 1,2-dibromoethanereactions is not the cleavage of the carbon-halogen bond, but rather theion release out of the active site.

Bioremediation

The present invention provides a number of dehalogenase enzymes usefulin bioremediation having improved enzymatic characteristics. Thepolynucleotides and polynucleotide products of the invention are usefulin, for example, groundwater treatment involving transformed host cellscontaining a polynucleotide or polypeptide of the invention (e.g., thebacteria Xanthobacter autotrophicus) and the haloalkane1,2-dichlorethane as well as removal of polychlorinated biphenyls(PCB's) from soil sediment.

The haloalkane dehalogenase of the invention are useful in carbon-halidereduction efforts. The enzymes of the invention initiate the degradationof haloalkanes. Alternatively, host cells containing a dehalogenasepolynucleotide or polypeptide of the invention can feed on thehaloalkanes and produce the detoxifying enzyme.

DEFINITIONS

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin. In oneembodiment, a “nucleic acid sequence” of the invention includes, forexample, a sequence encoding a polypeptide as set forth in Group B aminoacid sequences and variants thereof. In another embodiment, a “nucleicacid sequence” of the invention includes, for example, a sequence as setforth in Group A nucleic acid sequences, sequences complementarythereto, fragments of the foregoing sequences and variants thereof.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons).

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules. In one embodiment, an “amino acidsequence” or “polypeptide sequence” of the invention includes, forexample, a sequence as set forth in Group B amino acid sequences,fragments of the foregoing sequences and variants thereof. In anotherembodiment, an “amino acid sequence” of the invention includes, forexample, a sequence encoded by a polynucleotide having a sequence as setforth in Group B nucleic acid sequences, sequences complementarythereto, fragments of the foregoing sequences and variants thereof.

The term “polypeptide” as used herein, refers to amino acids joined toeach other by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Modificationscan occur anywhere in the polypeptide, including the peptide backbone,the amino acid side-chains and the amino or carboxyl termini. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide. Also agiven polypeptide may have many types of modifications. Modificationsinclude acetylation, acylation, ADP-ribosylation, amidation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment of aphosphatidylinositol, cross-linking cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristolyation, oxidation, PEGylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, and transfer-RNA mediated addition of aminoacids to protein such as arginylation. (See Creighton, T. E.,Proteins—Structure and Molecular Properties, 2nd Ed., W.H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)).

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 104-106 fold. However, the term “purified” also includes nucleicacids which have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders, and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids, and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone embodiment, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” polypeptides or protein are thoseprepared by chemical synthesis. Solid-phase chemical peptide synthesismethods can also be used to synthesize the polypeptide or fragments ofthe invention. Such method have been known in the art since the early1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (seealso Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2ndEd., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recentlybeen employed in commercially available laboratory peptide design andsynthesis kits (Cambridge Research Biochemicals). Such commerciallyavailable laboratory kits have generally utilized the teachings of H. M.Geysen, et al., Proc. Natl. Acad. Sci. USA, 81:3998 (1984) and providefor synthesizing peptides upon the tips of a multitude of “rods” or“pins” all of which are connected to a single plate. When such a systemis utilized, a plate of rods or pins is inverted and inserted into asecond plate of corresponding wells or reservoirs, which containsolutions for attaching or anchoring an appropriate amino acid to thepin's or rod's tips. By repeating such a process step, i.e., invertingand inserting the rod's and pin's tips into appropriate solutions, aminoacids are built into desired peptides. In addition, a number ofavailable FMOC peptide synthesis systems are available. For example,assembly of a polypeptide or fragment can be carried out on a solidsupport using an Applied Biosystems, Inc. Model 431A automated peptidesynthesizer. Such equipment provides ready access to the peptides of theinvention, either by direct synthesis or by synthesis of a series offragments that can be coupled using other known techniques.

A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

“Oligonucleotide” refers to either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, and in some aspects 90-95% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the known sequence comparisonalgorithms or by visual inspection. Typically, the substantial identityexists over a region of at least about 100 residues, and most commonlythe sequences are substantially identical over at least about 150-200residues. In some embodiments, the sequences are substantially identicalover the entire length of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an dehalogenase polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for dehalogenasebiological activity can be removed. Modified polypeptide sequences ofthe invention can be assayed for dehalogenase biological activity by anynumber of methods, including contacting the modified polypeptidesequence with an dehalogenase substrate and determining whether themodified polypeptide decreases the amount of specific substrate in theassay or increases the bioproducts of the enzymatic reaction of afunctional dehalogenase polypeptide with the substrate.

“Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In general two amino acid sequences are“substantially the same” or “substantially homologous” if they are atleast about 85% identical. Fragments which have different threedimensional structures as the naturally occurring protein are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature, andare well known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 n/ml sheared and denatured salmon sperm DNA. Hybridization couldoccur under reduced stringency conditions as described above, but in 35%formamide at a reduced temperature of 35° C. The temperature rangecorresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “variant” refers to polynucleotides or polypeptides of theinvention modified at one or more base pairs, codons, introns, exons, oramino acid residues (respectively) yet still retain the biologicalactivity of an dehalogenase of the invention. The polynucleotides orpolypeptides of the invention may also be modified by introduction of amodified base, such as inosine. Additionally, the modifications may,optionally, be repeated one or more times. Variants can be produced byany number of means including methods such as, for example, error-pronePCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,recursive ensemble mutagenesis, exponential ensemble mutagenesis,site-specific mutagenesis, gene reassembly, GSSM and any combination,permutation or iterative process thereof.

Enzymes are highly selective catalysts. Their hallmark is the ability tocatalyze reactions with exquisite stereo-, regio-, andchemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, enzymes are remarkably versatile. They can betailored to function in organic solvents, operate at extreme pHs (forexample, high pHs and low pHs) extreme temperatures (for example, hightemperatures and low temperatures), extreme salinity levels (forexample, high salinity and low salinity), and catalyze reactions withcompounds that are structurally unrelated to their natural,physiological substrates.

Enzymes are reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Moreover, unlike traditional chemical catalysts, enzymes arehighly enantio- and regio-selective. The high degree of functional groupspecificity exhibited by enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes are also capable of catalyzing many diverse reactions unrelatedto their physiological function in nature. For example, peroxidasescatalyze the oxidation of phenols by hydrogen peroxide. Peroxidases canalso catalyze hydroxylation reactions that are not related to the nativefunction of the enzyme. Other examples are proteases which catalyze thebreakdown of polypeptides. In organic solution some proteases can alsoacylate sugars, a function unrelated to the native function of theseenzymes.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds.

Each biocatalyst is specific for one functional group, or severalrelated functional groups, and can react with many starting compoundscontaining this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original compoundcan be produced with each iteration of biocatalytic derivitization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods. (For further teachings on modification ofmolecules, including small molecules, see PCT/US94/09174, hereinincorporated by reference in its entirety).

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly, that is somewhat related to stochasticshuffling, save that the nucleic acid building blocks are not shuffledor concatenated or chimerized randomly, but rather are assemblednon-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends, and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one embodiment of the invention, theannealed building pieces are treated with an enzyme, such as a ligase(e.g., T4 DNA ligase) to achieve covalent bonding of the buildingpieces.

In a another embodiment, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e., chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thedehalogenases of the present invention can be mutagenized in accordancewith the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofGroup A nucleic acid sequences) are aligned in order to select one ormore demarcation points, which demarcation points can be located at anarea of homology. The demarcation points can be used to delineate theboundaries of nucleic acid building blocks to be generated. Thus, thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the progenymolecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates, and preferably at almost all of theprogenitor templates. Even more preferably still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one embodiment, the gene reassembly process is performedexhaustively in order to generate an exhaustive library. In other words,all possible ordered combinations of the nucleic acid building blocksare represented in the set of finalized chimeric nucleic acid molecules.At the same time, the assembly order (i.e., the order of assembly ofeach building block in the 5′ to 3 sequence of each finalized chimericnucleic acid) in each combination is by design (or non-stochastic).Because of the non-stochastic nature of the method, the possibility ofunwanted side products is greatly reduced.

In another embodiment, the method provides that the gene reassemblyprocess is performed systematically, for example to generate asystematically compartmentalized library, with compartments that can bescreened systematically, e.g., one by one. In other words the inventionprovides that, through the selective and judicious use of specificnucleic acid building blocks, coupled with the selective and judicioususe of sequentially stepped assembly reactions, an experimental designcan be achieved where specific sets of progeny products are made in eachof several reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated preferably comprise a library of finalizedchimeric nucleic acid molecules having an overall assembly order that ischosen by design. In a particularly embodiment, such a generated libraryis comprised of greater than 10³ to greater than 10¹⁰⁰⁰ differentprogeny molecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one embodiment, this polynucleotide is a gene,which may be a man-made gene. According to another embodiment, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g., by mutagenesis) or in an in vivoprocess (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another embodiment, the invention provides that anucleic acid building block can be used to introduce an intron. Thus,the invention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). Preferably, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a preferredinstance, the recombination is facilitated by, or occurs at, areas ofhomology between the man-made, intron-containing gene and a nucleicacid, which serves as a recombination partner. In a particularlypreferred instance, the recombination partner may also be a nucleic acidgenerated by the invention, including a man-made gene or a man-made genepathway. Recombination may be facilitated by or may occur at areas ofhomology that exist at the one (or more) artificially introducedintron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which preferably hastwo ligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e., each having an overhang of zeronucleotides), or preferably one blunt end and one overhang, or morepreferably still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

According to one preferred embodiment, a nucleic acid building block isgenerated by chemical synthesis of two single-stranded nucleic acids(also referred to as single-stranded oligos) and contacting them so asto allow them to anneal to form a double-stranded nucleic acid buildingblock.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Preferredsizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherpreferred size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between), and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one embodiment, a double-stranded nucleic acid buildingblock is generated by first generating two single stranded nucleic acidsand allowing them to anneal to form a double-stranded nucleic acidbuilding block. The two strands of a double-stranded nucleic acidbuilding block may be complementary at every nucleotide apart from anythat form an overhang; thus containing no mismatches, apart from anyoverhang(s). According to another embodiment, the two strands of adouble-stranded nucleic acid building block are complementary at fewerthan every nucleotide apart from any that form an overhang. Thus,according to this embodiment, a double-stranded nucleic acid buildingblock can be used to introduce codon degeneracy. Preferably the codondegeneracy is introduced using the site-saturation mutagenesis describedherein, using one or more N,N,G/T cassettes or alternatively using oneor more N,N,N cassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

In vivo shuffling of molecules is useful in providing variants and canbe performed utilizing the natural property of cells to recombinemultimers. While recombination in vivo has provided the major naturalroute to molecular diversity, genetic recombination remains a relativelycomplex process that involves 1) the recognition of homologies; 2)strand cleavage, strand invasion, and metabolic steps leading to theproduction of recombinant chiasma; and finally 3) the resolution ofchiasma into discrete recombined molecules. The formation of the chiasmarequires the recognition of homologous sequences.

In another embodiment, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 43, 45, 47, and combinations thereof) into asuitable host cell. The regions of partial sequence homology promoteprocesses which result in sequence reorganization producing a hybridpolynucleotide. The term “hybrid polynucleotide,” as used herein, is anynucleotide sequence which results from the method of the presentinvention and contains sequence from at least two originalpolynucleotide sequences. Such hybrid polynucleotides can result fromintermolecular recombination events which promote sequence integrationbetween DNA molecules. In addition, such hybrid polynucleotides canresult from intramolecular reductive reassortment processes whichutilize repeated sequences to alter a nucleotide sequence within a DNAmolecule.

The invention provides a means for generating hybrid polynucleotideswhich may encode biologically active hybrid polypeptides (e.g., hybridhaloalkane dehalogenase). In one aspect, the original polynucleotidesencode biologically active polypeptides. The method of the inventionproduces new hybrid polypeptides by utilizing cellular processes whichintegrate the sequence of the original polynucleotides such that theresulting hybrid polynucleotide encodes a polypeptide demonstratingactivities derived from the original biologically active polypeptides.For example, the original polynucleotides may encode a particular enzymefrom different microorganisms. An enzyme encoded by a firstpolynucleotide from one organism or variant may, for example, functioneffectively under a particular environmental condition, e.g., highsalinity. An enzyme encoded by a second polynucleotide from a differentorganism or variant may function effectively under a differentenvironmental condition, such as extremely high temperatures. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme which exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide may function effectively underenvironmental conditions shared by each of the enzymes encoded by thefirst and second polynucleotides, e.g., high salinity and extremetemperatures.

Enzymes encoded by the polynucleotides of the invention include, but arenot limited to, hydrolases, dehalogenases and haloalkane dehalogenases.A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding hydrolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized hydrolase activities obtained from each of theoriginal enzymes, i.e., the type of bond on which the hydrolase acts andthe temperature at which the hydrolase functions. Thus, for example, thehydrolase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid hydrolase from the original hydrolases,such as: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds,i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, forexample, the temperature, pH or salt concentration at which the hybridpolypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria, andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms are particularly preferred. Such enzymes may function attemperatures above 100° C. in terrestrial hot springs and deep seathermal vents, at temperatures below 0° C. in arctic waters, in thesaturated salt environment of the Dead Sea, at pH values around 0 incoal deposits and geothermal sulfur-rich springs, or at pH valuesgreater than 11 in sewage sludge. For example, several esterases andlipases cloned and expressed from extremophilic organisms show highactivity throughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis, et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981), and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer, and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

Host cells containing the polynucleotides of interest can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan. The clones which are identified ashaving the specified enzyme activity may then be sequenced to identifythe polynucleotide sequence encoding an enzyme having the enhancedactivity.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides. Polyketides are molecules which are anextremely rich source of bioactivities, including antibiotics (such astetracyclines and erythromycin), anti-cancer agents (daunomycin),immunosuppressants (FK506 and rapamycin), and veterinary products(monensin). Many polyketides (produced by polyketide synthases) arevaluable as therapeutic agents. Polyketide synthases are multifunctionalenzymes that catalyze the biosynthesis of an enormous variety of carbonchains differing in length and patterns of functionality andcyclization. Polyketide synthase genes fall into gene clusters and atleast one type (designated type I) of polyketide synthases have largesize genes and enzymes, complicating genetic manipulation and in vitrostudies of these genes/proteins.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affect high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Aparticularly preferred embodiment is to use cloning vectors, referred toas “fosmids” or bacterial artificial chromosome (BAC) vectors. These arederived from E. coli f-factor which is able to stably integrate largesegments of genomic DNA. When integrated with DNA from a mixeduncultured environmental sample, this makes it possible to achieve largegenomic fragments in the form of a stable “environmental DNA library.”Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated intoan appropriate vector, two or more vectors containing differentpolyketide synthase gene clusters can be introduced into a suitable hostcell. Regions of partial sequence homology shared by the gene clusterswill promote processes which result in sequence reorganization resultingin a hybrid gene cluster. The novel hybrid gene cluster can then bescreened for enhanced activities not found in the original geneclusters.

Therefore, in a one embodiment, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, said at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

As representative examples of expression vectors which may be used,there may be mentioned viral particles, baculovirus, phage, plasmids,phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies andderivatives of SV40), P1-based artificial chromosomes, yeast plasmids,yeast artificial chromosomes, and any other vectors specific forspecific hosts of interest (such as bacillus, Aspergillus and yeast).Thus, for example, the DNA may be included in any one of a variety ofexpression vectors for expressing a polypeptide. Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. The following vectors are provided by way ofexample: Bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNHvectors, (lambda-ZAP vectors (Stratagene)); ptrc99a, pKK223-3, pDR540,pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV,pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vectormay be used so long as they are replicable and viable in the host. Lowcopy number or high copy number vectors may be employed with the presentinvention.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus, and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular,” RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector, and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences, and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals, and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNAseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences, and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

-   -   1) The use of vectors only stably maintained when the construct        is reduced in complexity.    -   2) The physical recovery of shortened vectors by physical        procedures. In this case, the cloning vector would be recovered        using standard plasmid isolation procedures and size        fractionated on either an agarose gel, or column with a low        molecular weight cut off utilizing standard procedures.    -   3) The recovery of vectors containing interrupted genes which        can be selected when insert size decreases.    -   4) The use of direct selection techniques with an expression        vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector, and transfected into an appropriate host cell. The cells arethen propagated and “reductive reassortment” is effected. The rate ofthe reductive reassortment process may be stimulated by the introductionof DNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

Optionally, the method comprises the additional step of screening thelibrary members of the shuffled pool to identify individual shuffledlibrary members having the ability to bind or otherwise interact, orcatalyze a particular reaction (e.g., such as catalytic domain of anenzyme) with a predetermined macromolecule, such as for example aproteinaceous receptor, an oligosaccharide, virion, or otherpredetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution, and the like),and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine (SeeSun and Hurley, (1992)); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See, for example, van de Poll, et al. (1992)); or an N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll, et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[α]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”), andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Especially preferred means for slowing or haltingPCR amplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (gene site saturation mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence, and preferably but notnecessarily a second homologous sequence. The downstream progenytranslational products from the use of such oligos include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,N sequence includes codons for all 20amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions, and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e., a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g., in anoligo) a degenerate triplet sequence comprised of only one N, where saidN can be in the first second or third position of the triplet. Any otherbases including any combinations and permutations thereof can be used inthe remaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan optionally be used in combination with degenerate primers disclosed.It is appreciated that in some situations, it is advantageous to usenondegenerate oligos to generate specific point mutations in a workingpolynucleotide. This provides a means to generate specific silent pointmutations, point mutations leading to corresponding amino acid changes,and point mutations that cause the generation of stop codons and thecorresponding expression of polypeptide fragments.

Thus, in a preferred embodiment of this invention, each saturationmutagenesis reaction vessel contains polynucleotides encoding at least20 progeny polypeptide molecules such that all 20 amino acids arerepresented at the one specific amino acid position corresponding to thecodon position mutagenized in the parental polynucleotide. The 32-folddegenerate progeny polypeptides generated from each saturationmutagenesis reaction vessel can be subjected to clonal amplification(e.g., cloned into a suitable E. coli host using an expression vector)and subjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid, and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined—6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is preferably everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(preferably a subset totaling from 15 to 100,000) to mutagenesis.Preferably, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations are preferablyintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Preferred cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette ispreferably about 1-500 bases in length) in defined polynucleotidesequence to be mutagenized (wherein the sequence to be mutagenized ispreferably from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons, and groupingsof particular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF), and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence, and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In a particularly preferred exemplification a grouping of mutations thatcan be introduced into a mutagenic cassette, this invention specificallyprovides for degenerate codon substitutions (using degenerate oligos)that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, and 20 amino acids at each position, and a library ofpolypeptides encoded thereby.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of Group A nucleic acid sequences, and sequencessubstantially identical thereto, the sequences complementary thereto, ora fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,150, 200, 300, 400, or 500 consecutive bases of one of the sequences ofa Group A nucleic acid sequence (or the sequences complementarythereto). The isolated, nucleic acids may comprise DNA, including cDNA,genomic DNA, and synthetic DNA. The DNA may be double-stranded orsingle-stranded, and if single stranded may be the coding strand ornon-coding (anti-sense) strand. Alternatively, the isolated nucleicacids may comprise RNA.

As discussed in more detail below, the isolated nucleic acids of one ofthe Group A nucleic acid sequences, and sequences substantiallyidentical thereto, may be used to prepare one of the polypeptides of aGroup B amino acid sequence, and sequences substantially identicalthereto, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40,50, 75, 100, or 150 consecutive amino acids of one of the polypeptidesof Group B amino acid sequences, and sequences substantially identicalthereto.

Accordingly, another aspect of the invention is an isolated nucleic acidwhich encodes one of the polypeptides of Group B amino acid sequences,and sequences substantially identical thereto, or fragments comprisingat least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids of one of the polypeptides of the Group B amino acidsequences. The coding sequences of these nucleic acids may be identicalto one of the coding sequences of one of the nucleic acids of Group Anucleic acid sequences, or a fragment thereof or may be different codingsequences which encode one of the polypeptides of Group B amino acidsequences, sequences substantially identical thereto, and fragmentshaving at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of Group B amino acidsequences, as a result of the redundancy or degeneracy of the geneticcode. The genetic code is well known to those of skill in the art andcan be obtained, for example, on page 214 of B. Lewin, Genes VI, OxfordUniversity Press, 1997, the disclosure of which is incorporated hereinby reference.

The isolated nucleic acid which encodes one of the polypeptides of GroupB amino acid sequences, and sequences substantially identical thereto,may include, but is not limited to: only the coding sequence of one ofGroup A nucleic acid sequences, and sequences substantially identicalthereto, and additional coding sequences, such as leader sequences orproprotein sequences and non-coding sequences, such as introns ornon-coding sequences 5′ and/or 3′ of the coding sequence. Thus, as usedherein, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only the coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

Alternatively, the nucleic acid sequences of Group A nucleic acidsequences, and sequences substantially identical thereto, may bemutagenized using conventional techniques, such as site directedmutagenesis, or other techniques familiar to those skilled in the art,to introduce silent changes into the polynucleotides of Group A nucleicacid sequences, and sequences substantially identical thereto. As usedherein, “silent changes” include, for example, changes which do notalter the amino acid sequence encoded by the polynucleotide. Suchchanges may be desirable in order to increase the level of thepolypeptide produced by host cells containing a vector encoding thepolypeptide by introducing codons or codon pairs which occur frequentlyin the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of Group B amino acidsequences, and sequences substantially identical thereto. Suchnucleotide changes may be introduced using techniques such as sitedirected mutagenesis, random chemical mutagenesis, exonuclease IIIdeletion, and other recombinant DNA techniques. Alternatively, suchnucleotide changes may be naturally occurring allelic variants which areisolated by identifying nucleic acids which specifically hybridize toprobes comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,200, 300, 400, or 500 consecutive bases of one of the sequences of GroupA nucleic acid sequences, and sequences substantially identical thereto(or the sequences complementary thereto) under conditions of high,moderate, or low stringency as provided herein.

The isolated nucleic acids of Group A nucleic acid sequences, andsequences substantially identical thereto, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of Group A nucleic acid sequences, and sequences substantiallyidentical thereto, or the sequences complementary thereto may also beused as probes to determine whether a biological sample, such as a soilsample, contains an organism having a nucleic acid sequence of theinvention or an organism from which the nucleic acid was obtained. Insuch procedures, a biological sample potentially harboring the organismfrom which the nucleic acid was isolated is obtained and nucleic acidsare obtained from the sample. The nucleic acids are contacted with theprobe under conditions which permit the probe to specifically hybridizeto any complementary sequences from which are present therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel, et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (1997) and Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989), the entire disclosures of which areincorporated herein by reference.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one embodiment, the amplification reaction maycomprise a PCR reaction. PCR protocols are described in Ausubel andSambrook, supra. Alternatively, the amplification may comprise a ligasechain reaction, 3SR, or strand displacement reaction. (See Barany, F.,“The Ligase Chain Reaction in a PCR World”, PCR Methods andApplications, 1:5-16, 1991; Fahy, E., et al., “Self-sustained SequenceReplication (3SR): An Isothermal Transcription-based AmplificationSystem Alternative to PCR”, PCR Methods and Applications, 1:25-33, 1991;and Walker, G. T., et al., “Strand Displacement Amplification-anIsothermal in vitro DNA Amplification Technique”, Nucleic Acid Research20:1691-1696, 1992, the disclosures of which are incorporated herein byreference in their entireties). In such procedures, the nucleic acids inthe sample are contacted with the probes, the amplification reaction isperformed, and any resulting amplification product is detected. Theamplification product may be detected by performing gel electrophoresison the reaction products and staining the gel with an intercalator suchas ethidium bromide. Alternatively, one or more of the probes may belabeled with a radioactive isotope and the presence of a radioactiveamplification product may be detected by autoradiography after gelelectrophoresis.

Probes derived from sequences near the ends of the sequences of Group Anucleic acid sequences, and sequences substantially identical thereto,may also be used in chromosome walking procedures to identify clonescontaining genomic sequences located adjacent to the sequences of GroupA nucleic acid sequences, and sequences substantially identical thereto.Such methods allow the isolation of genes which encode additionalproteins from the host organism.

The isolated nucleic acids of Group A nucleic acid sequences, andsequences substantially identical thereto, the sequences complementarythereto, or a fragment comprising at least 10, 15, 20, 25, 30, 35, 40,50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of one of thesequences of Group A nucleic acid sequences, and sequences substantiallyidentical thereto, or the sequences complementary thereto may be used asprobes to identify and isolate related nucleic acids. In someembodiments, the related nucleic acids may be cDNAs or genomic DNAs fromorganisms other than the one from which the nucleic acid was isolated.For example, the other organisms may be related organisms. In suchprocedures, a nucleic acid sample is contacted with the probe underconditions which permit the probe to specifically hybridize to relatedsequences. Hybridization of the probe to nucleic acids from the relatedorganism is then detected using any of the methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt'sreagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50%formamide. The formulas for SSC and Denhardt's solutions are listed inSambrook, et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). Typically, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

For example, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55% or at least 50% homology to a nucleic acidsequence selected from the group consisting of one of the sequences ofGroup A nucleic acid sequences, and sequences substantially identicalthereto, or fragments comprising at least about 10, 15, 20, 25, 30, 35,40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases thereof,and the sequences complementary thereto. Homology may be measured usingthe alignment algorithm. For example, the homologous polynucleotides mayhave a coding sequence which is a naturally occurring allelic variant ofone of the coding sequences described herein. Such allelic variants mayhave a substitution, deletion or addition of one or more nucleotideswhen compared to the nucleic acids of Group A nucleic acid sequences orthe sequences complementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55% or at least 50% homology to a polypeptide havingthe sequence of one of Group B amino acid sequences, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof as determined using a sequence alignment algorithm (e.g., suchas the FASTA version 3.0t78 algorithm with the default parameters).

Another aspect of the invention is an isolated or purified polypeptidecomprising the sequence of one of Group A nucleic acid sequences, andsequences substantially identical thereto, or fragments comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. As discussed above, such polypeptidesmay be obtained by inserting a nucleic acid encoding the polypeptideinto a vector such that the coding sequence is operably linked to asequence capable of driving the expression of the encoded polypeptide ina suitable host cell. For example, the expression vector may comprise apromoter, a ribosome binding site for translation initiation and atranscription terminator. The vector may also include appropriatesequences for amplifying expression.

Promoters suitable for expressing the polypeptide or fragment thereof inbacteria include the E. coli lac or trp promoters, the lacI promoter,the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter,the lambda P_(R) promoter, the lambda P_(L) promoter, promoters fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), and the acid phosphatase promoter. Fungal promoters include the ∀factor promoter. Eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, heat shock promoters, theearly and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences, and 5′flanking nontranscribed sequences. In some embodiments, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli, and the S. cerevisiae TRP1 gene.

After the expression libraries have been generated one can include theadditional step of “biopanning” such libraries prior to screening bycell sorting. The “biopanning” procedure refers to a process foridentifying clones having a specified biological activity by screeningfor sequence homology in a library of clones prepared by (i) selectivelyisolating target DNA, from DNA derived from at least one microorganism,by use of at least one probe DNA comprising at least a portion of a DNAsequence encoding an biological having the specified biologicalactivity; and (ii) optionally transforming a host with isolated targetDNA to produce a library of clones which are screened for the specifiedbiological activity.

The probe DNA used for selectively isolating the target DNA of interestfrom the DNA derived from at least one microorganism can be afull-length coding region sequence or a partial coding region sequenceof DNA for an enzyme of known activity. The original DNA library can bepreferably probed using mixtures of probes comprising at least a portionof the DNA sequence encoding an enzyme having the specified enzymeactivity. These probes or probe libraries are preferably single-strandedand the microbial DNA which is probed has preferably been converted intosingle-stranded form. The probes that are particularly suitable arethose derived from DNA encoding enzymes having an activity similar oridentical to the specified enzyme activity which is to be screened.

The probe DNA should be at least about 10 bases and preferably at least15 bases. In one embodiment, the entire coding region may be employed asa probe. Conditions for the hybridization in which target DNA isselectively isolated by the use of at least one DNA probe will bedesigned to provide a hybridization stringency of at least about 50%sequence identity, more particularly a stringency providing for asequence identity of at least about 70%.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42° C. (moderate stringency conditions); and0.1×SSC at about 68° C. (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

Hybridization techniques for probing a microbial DNA library to isolatetarget DNA of potential interest are well known in the art and any ofthose which are described in the literature are suitable for use herein,particularly those which use a solid phase-bound, directly or indirectlybound, probe DNA for ease in separation from the remainder of the DNAderived from the microorganisms.

Preferably the probe DNA is “labeled” with one partner of a specificbinding pair (i.e., a ligand) and the other partner of the pair is boundto a solid matrix to provide ease of separation of target from itssource. The ligand and specific binding partner can be selected from, ineither orientation, the following: (1) an antigen or hapten and anantibody or specific binding fragment thereof; (2) biotin or iminobiotinand avidin or streptavidin; (3) a sugar and a lectin specific therefor;(4) an enzyme and an inhibitor therefor; (5) an apoenzyme and cofactor;(6) complementary homopolymeric oligonucleotides; and (7) a hormone anda receptor therefor. The solid phase is preferably selected from: (1) aglass or polymeric surface; (2) a packed column of polymeric beads; and(3) magnetic or paramagnetic particles.

Further, it is optional but desirable to perform an amplification of thetarget DNA that has been isolated. In this embodiment the target DNA isseparated from the probe DNA after isolation. It is then amplifiedbefore being used to transform hosts. The double stranded DNA selectedto include as at least a portion thereof a predetermined DNA sequencecan be rendered single stranded, subjected to amplification andreannealed to provide amplified numbers of selected double stranded DNA.Numerous amplification methodologies are now well known in the art.

The selected DNA is then used for preparing a library for screening bytransforming a suitable organism. Hosts, particularly those specificallyidentified herein as preferred, are transformed by artificialintroduction of the vectors containing the target DNA by inoculationunder conditions conducive for such transformation.

The resultant libraries of transformed clones are then screened forclones which display activity for the enzyme of interest.

Having prepared a multiplicity of clones from DNA selectively isolatedfrom an organism, such clones are screened for a specific enzymeactivity and to identify the clones having the specified enzymecharacteristics.

The screening for enzyme activity may be effected on individualexpression clones or may be initially effected on a mixture ofexpression clones to ascertain whether or not the mixture has one ormore specified enzyme activities. If the mixture has a specified enzymeactivity, then the individual clones may be rescreened utilizing a FACSmachine for such enzyme activity or for a more specific activity.Alternatively, encapsulation techniques such as gel microdroplets, maybe employed to localize multiple clones in one location to be screenedon a FACS machine for positive expressing clones within the group ofclones which can then be broken out into individual clones to bescreened again on a FACS machine to identify positive individual clones.Thus, for example, if a clone mixture has hydrolase activity, then theindividual clones may be recovered and screened utilizing a FACS machineto determine which of such clones has hydrolase activity. As usedherein, “small insert library” means a gene library containing cloneswith random small size nucleic acid inserts of up to approximately 5000base pairs. As used herein, “large insert library” means a gene librarycontaining clones with random large size nucleic acid inserts ofapproximately 5000 up to several hundred thousand base pairs or greater.

As described with respect to one of the above aspects, the inventionprovides a process for enzyme activity screening of clones containingselected DNA derived from a microorganism which process includes:screening a library for specified enzyme activity, said libraryincluding a plurality of clones, said clones having been prepared byrecovering from genomic DNA of a microorganism selected DNA, which DNAis selected by hybridization to at least one DNA sequence which is allor a portion of a DNA sequence encoding an enzyme having the specifiedactivity; and transforming a host with the selected DNA to produceclones which are screened for the specified enzyme activity.

In one embodiment, a DNA library derived from a microorganism issubjected to a selection procedure to select therefrom DNA whichhybridizes to one or more probe DNA sequences which is all or a portionof a DNA sequence encoding an enzyme having the specified enzymeactivity by:

-   -   (a) rendering the double-stranded genomic DNA population into a        single-stranded DNA population;    -   (b) contacting the single-stranded DNA population of (a) with        the DNA probe bound to a ligand under conditions permissive of        hybridization so as to produce a double-stranded complex of        probe and members of the genomic DNA population which hybridize        thereto;    -   (c) contacting the double-stranded complex of (b) with a solid        phase specific binding partner for said ligand so as to produce        a solid phase complex;    -   (d) separating the solid phase complex from the single-stranded        DNA population of (b);    -   (e) releasing from the probe the members of the genomic        population which had bound to the solid phase bound probe;    -   (f) forming double-stranded DNA from the members of the genomic        population of (e);    -   (g) introducing the double-stranded DNA of (f) into a suitable        host to form a library containing a plurality of clones        containing the selected DNA; and    -   (h) screening the library for the specified enzyme activity.

In another aspect, the process includes a preselection to recover DNAincluding signal or secretion sequences. In this manner it is possibleto select from the genomic DNA population by hybridization ashereinabove described only DNA which includes a signal or secretionsequence. The following paragraphs describe the protocol for thisembodiment of the invention, the nature and function of secretion signalsequences in general and a specific exemplary application of suchsequences to an assay or selection process.

One aspect further comprises, after (a) but before (b) above, the stepsof:

-   -   (ai) contacting the single-stranded DNA population of (a) with a        ligand-bound oligonucleotide probe that is complementary to a        secretion signal sequence unique to a given class of proteins        under conditions permissive of hybridization to form a        double-stranded complex;    -   (aii) contacting the double-stranded complex of (ai) with a        solid phase specific binding partner for said ligand so as to        produce a solid phase complex;    -   (aiii) separating the solid phase complex from the        single-stranded DNA population of (a);    -   (aiv) releasing the members of the genomic population which had        bound to said solid phase bound probe; and    -   (av) separating the solid phase bound probe from the members of        the genomic population which had bound thereto.

The DNA which has been selected and isolated to include a signalsequence is then subjected to the selection procedure hereinabovedescribed to select and isolate therefrom DNA which binds to one or moreprobe DNA sequences derived from DNA encoding an enzyme(s) having thespecified enzyme activity. This procedure is described and exemplifiedin U.S. Ser. No. 08/692,002, filed Aug. 2, 1996, incorporated herein byreference.

In vivo biopanning may be performed utilizing a FACS-based machine.Complex gene libraries are constructed with vectors which containelements which stabilize transcribed RNA. For example, the inclusion ofsequences which result in secondary structures such as hairpins whichare designed to flank the transcribed regions of the RNA would serve toenhance their stability, thus increasing their half life within thecell. The probe molecules used in the biopanning process consist ofoligonucleotides labeled with reporter molecules that only fluoresceupon binding of the probe to a target molecule. These probes areintroduced into the recombinant cells from the library using one ofseveral transformation methods. The probe molecules bind to thetranscribed target mRNA resulting in DNA/RNA heteroduplex molecules.Binding of the probe to a target will yield a fluorescent signal whichis detected and sorted by the FACS machine during the screening process.

In some embodiments, the nucleic acid encoding one of the polypeptidesof Group B amino acid sequences, and sequences substantially identicalthereto, or fragments comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is assembledin appropriate phase with a leader sequence capable of directingsecretion of the translated polypeptide or fragment thereof. Optionally,the nucleic acid can encode a fusion polypeptide in which one of thepolypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof is fused to heterologous peptides or polypeptides, such asN-terminal identification peptides which impart desired characteristics,such as increased stability or simplified purification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel, et al., Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook, et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989), the entire disclosures of which areincorporated herein by reference. Such procedures and others are deemedto be within the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989), the disclosure of which ishereby incorporated by reference.

Particular bacterial vectors which may be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Salmonella typhimurium and variousspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus,fungal cells, such as yeast, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma, andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981), and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of Group B amino acid sequences, andsequences substantially identical thereto, or fragments comprising atleast 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof can be synthetically produced by conventionalpeptide synthesizers. In other embodiments, fragments or portions of thepolypeptides may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof using mRNAs transcribed from a DNA construct comprising apromoter operably linked to a nucleic acid encoding the polypeptide orfragment thereof. In some embodiments, the DNA construct may belinearized prior to conducting an in vitro transcription reaction. Thetranscribed mRNA is then incubated with an appropriate cell-freetranslation extract, such as a rabbit reticulocyte extract, to producethe desired polypeptide or fragment thereof.

The invention also relates to variants of the polypeptides of Group Bamino acid sequences, and sequences substantially identical thereto, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof. The term “variant” includesderivatives or analogs of these polypeptides. In particular, thevariants may differ in amino acid sequence from the polypeptides ofGroup B amino acid sequences, and sequences substantially identicalthereto, by one or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

The variants may be naturally occurring or created in vitro. Inparticular, such variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures.

Other methods of making variants are also familiar to those skilled inthe art. These include procedures in which nucleic acid sequencesobtained from natural isolates are modified to generate nucleic acidswhich encode polypeptides having characteristics which enhance theirvalue in industrial or laboratory applications. In such procedures, alarge number of variant sequences having one or more nucleotidedifferences with respect to the sequence obtained from the naturalisolate are generated and characterized. Typically, these nucleotidedifferences result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described in Leung, D. W., et al., Technique, 1:11-15, 1989) andCaldwell, R. C. & Joyce, G. F., PCR Methods Applic., 2:28-33, 1992, thedisclosure of which is incorporated herein by reference in its entirety.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in Reidhaar-Olson, J. F. &Sauer, R. T., et al., Science, 241:53-57, 1988, the disclosure of whichis incorporated herein by reference in its entirety. Briefly, in suchprocedures a plurality of double stranded oligonucleotides bearing oneor more mutations to be introduced into the cloned DNA are synthesizedand inserted into the cloned DNA to be mutagenized. Clones containingthe mutagenized DNA are recovered and the activities of the polypeptidesthey encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in U.S. Pat. No.5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassembly byInterrupting Synthesis”, the disclosure of which is incorporated hereinby reference in its entirety.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described inStemmer, W. P., PNAS, USA, 91:10747-10751, 1994, the disclosure of whichis incorporated herein by reference. Briefly, in such procedures aplurality of nucleic acids to be recombined are digested with DNase togenerate fragments having an average size of 50-200 nucleotides.Fragments of the desired average size are purified and resuspended in aPCR mixture. PCR is conducted under conditions which facilitaterecombination between the nucleic acid fragments. For example, PCR maybe performed by resuspending the purified fragments at a concentrationof 10-30 ng/:1 in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl₂, 50 mMKCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5 units of Taqpolymerase per 100:1 of reaction mixture is added and PCR is performedusing the following regime: 94° C. for 60 seconds, 94° C. for 30seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45 times)and 72° C. for 5 minutes. However, it will be appreciated that theseparameters may be varied as appropriate. In some embodiments,oligonucleotides may be included in the PCR reactions. In otherembodiments, the Klenow fragment of DNA polymerase I may be used in afirst set of PCR reactions and Taq polymerase may be used in asubsequent set of PCR reactions. Recombinant sequences are isolated andthe activities of the polypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In someembodiments, random mutations in a sequence of interest are generated bypropagating the sequence of interest in a bacterial strain, such as anE. coli strain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations” the disclosure ofwhich is incorporated herein by reference in its entirety.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D. C., PNAS, USA,89:7811-7815, 1992, the disclosure of which is incorporated herein byreference in its entirety.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S, and Youvan, D. C., Biotechnology Research, 11:1548-1552,1993, the disclosure of which incorporated herein by reference in itsentirety. Random and site-directed mutagenesis are described in Arnold,F. H., Current Opinion in Biotechnology, 4:450-455, 1993, the disclosureof which is incorporated herein by reference in its entirety.

In some embodiments, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis”, and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis”, both of which are incorporated herein by reference.

The variants of the polypeptides of Group B amino acid sequences may bevariants in which one or more of the amino acid residues of thepolypeptides of the Group B amino acid sequences are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of the Group B amino acid sequences includes asubstituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide.

In some embodiments, the fragments, derivatives and analogs retain thesame biological function or activity as the polypeptides of Group Bamino acid sequences, and sequences substantially identical thereto. Inother embodiments, the fragment, derivative, or analog includes aproprotein, such that the fragment, derivative, or analog can beactivated by cleavage of the proprotein portion to produce an activepolypeptide.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or more than about 95% homology to one of the polypeptides of Group Bamino acid sequences, and sequences substantially identical thereto, ora fragment comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof. Homology may be determinedusing any of the programs described above which aligns the polypeptidesor fragments being compared and determines the extent of amino acididentity or similarity between them. It will be appreciated that aminoacid “homology” includes conservative amino acid substitutions such asthose described above.

The polypeptides or fragments having homology to one of the polypeptidesof Group B amino acid sequences, and sequences substantially identicalthereto, or a fragment comprising at least about 5, 10, 15, 20, 25, 30,35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may beobtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byproteolytic digestion, gel electrophoresis and/or microsequencing. Thesequence of the prospective homologous polypeptide or fragment can becompared to one of the polypeptides of Group B amino acid sequences, andsequences substantially identical thereto, or a fragment comprising atleast about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using any of the programs describedabove.

Another aspect of the invention is an assay for identifying fragments orvariants of Group B amino acid sequences, and sequences substantiallyidentical thereto, which retain the enzymatic function of thepolypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto. For example the fragments or variantsof said polypeptides, may be used to catalyze biochemical reactions,which indicate that the fragment or variant retains the enzymaticactivity of the polypeptides in the Group B amino acid sequences.

The assay for determining if fragments of variants retain the enzymaticactivity of the polypeptides of Group B amino acid sequences, andsequences substantially identical thereto includes the steps of:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction, and detecting either a decrease in the level of substrate oran increase in the level of the specific reaction product of thereaction between the polypeptide and substrate.

The polypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof may be used in a variety of applications. For example, thepolypeptides or fragments thereof may be used to catalyze biochemicalreactions. In accordance with one aspect of the invention, there isprovided a process for utilizing the polypeptides of Group B amino acidsequences, and sequences substantially identical thereto orpolynucleotides encoding such polypeptides for hydrolyzing glycosidiclinkages. In such procedures, a substance containing a glycosidiclinkage (e.g., a starch) is contacted with one of the polypeptides ofGroup B amino acid sequences, or sequences substantially identicalthereto under conditions which facilitate the hydrolysis of theglycosidic linkage.

The polypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof, may also be used to generate antibodies which bind specificallyto the polypeptides or fragments. The resulting antibodies may be usedin immunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of Group B amino acidsequences, and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of Group B aminoacid sequences, and sequences substantially identical thereto, orfragment thereof. After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of Group Bamino acid sequences, and sequences substantially identical thereto, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof can be obtained by directinjection of the polypeptides into an animal or by administering thepolypeptides to an animal, for example, a nonhuman. The antibody soobtained will then bind the polypeptide itself. In this manner, even asequence encoding only a fragment of the polypeptide can be used togenerate antibodies which may bind to the whole native polypeptide. Suchantibodies can then be used to isolate the polypeptide from cellsexpressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975, the disclosure of which is incorporated herein byreference), the trioma technique, the human B-cell hybridoma technique(Kozbor, et al., Immunology Today, 4:72, 1983, the disclosure of whichis incorporated herein by reference), and the EBV-hybridoma technique(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96, the disclosure of which is incorporated hereinby reference).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778, the disclosure of which is incorporated herein byreference) can be adapted to produce single chain antibodies to thepolypeptides of Group B amino acid sequences, and sequencessubstantially identical thereto, or fragments comprising at least 5, 10,15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof. Alternatively, transgenic mice may be used to express humanizedantibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of Group B amino acidsequences, and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof may be used in screening for similarpolypeptides from other organisms and samples. In such techniques,polypeptides from the organism are contacted with the antibody and thosepolypeptides which specifically bind the antibody are detected. Any ofthe procedures described above may be used to detect antibody binding.One such screening assay is described in “Methods for MeasuringCellulase Activities”, Methods in Enzymology, Vol 160, pp. 87-116, whichis hereby incorporated by reference in its entirety.

As used herein the term “nucleic acid sequence as set forth in SEQ IDNOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,43, 45 and 47” encompasses the nucleotide sequences of Group A nucleicacid sequences, and sequences substantially identical thereto, as wellas sequences homologous to Group A nucleic acid sequences, and fragmentsthereof and sequences complementary to all of the preceding sequences.The fragments include portions of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 43, 45 and 47 comprising atleast 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or500 consecutive nucleotides of Group A nucleic acid sequences, andsequences substantially identical thereto. Homologous sequences andfragments of Group A nucleic acid sequences, and sequences substantiallyidentical thereto, refer to a sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% homology tothese sequences. Homology may be determined using any of the computerprograms and parameters described herein, including FASTA version 3.0t78with the default parameters. Homologous sequences also include RNAsequences in which uridines replace the thymines in the nucleic acidsequences as set forth in the Group A nucleic acid sequences. Thehomologous sequences may be obtained using any of the proceduresdescribed herein or may result from the correction of a sequencingerror. It will be appreciated that the nucleic acid sequences as setforth in Group A nucleic acid sequences, and sequences substantiallyidentical thereto, can be represented in the traditional singlecharacter format (See the inside back cover of Stryer, Lubert,Biochemistry, 3rd Ed., W. H Freeman & Co., New York.) or in any otherformat which records the identity of the nucleotides in a sequence.

As used herein the term “a polypeptide sequence as set forth in SEQ IDNO's: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 44, 46 and 48” encompasses the polypeptide sequence of Group B aminoacid sequences, and sequences substantially identical thereto, which areencoded by a sequence as set forth in SEQ ID NO's: 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 43, 45 and 47,polypeptide sequences homologous to the polypeptides of Group B aminoacid sequences, and sequences substantially identical thereto, orfragments of any of the preceding sequences. Homologous polypeptidesequences refer to a polypeptide sequence having at least 99%, 98%, 97%,96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% homology to oneof the polypeptide sequences of the Group B amino acid sequences.Homology may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,or 150 consecutive amino acids of the polypeptides of Group B amino acidsequences, and sequences substantially identical thereto. It will beappreciated that the polypeptide codes as set forth in Group B aminoacid sequences, and sequences substantially identical thereto, can berepresented in the traditional single character format or three letterformat (See the inside back cover of Sayer, Lubert. Biochemistry, 3rdEd., W. H. Freeman & Co., New York.) or in any other format whichrelates the identity of the polypeptides in a sequence.

It will be appreciated by those skilled in the art that a nucleic acidsequence as set forth in SEQ ID NO's: 3, 5, 7, 9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29, 31, 33, 35, 37, 43, 45 and 47, and a polypeptidesequence as set forth in SEQ ID NO's: 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 44, 46 and 48 can be stored,recorded, and manipulated on any medium which can be read and accessedby a computer. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any of the presently known methods forrecording information on a computer readable medium to generatemanufactures comprising one or more of the nucleic acid sequences as setforth in Group A nucleic acid sequences, and sequences substantiallyidentical thereto, one or more of the polypeptide sequences as set forthin Group B amino acid sequences, and sequences substantially identicalthereto. Another aspect of the invention is a computer readable mediumhaving recorded thereon at least 2, 5, 10, 15, or 20 nucleic acidsequences as set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences as set forthin Group A nucleic acid sequences, and sequences substantially identicalthereto. Another aspect of the invention is a computer readable mediumhaving recorded thereon one or more of the polypeptide sequences as setforth in Group B amino acid sequences, and sequences substantiallyidentical thereto. Another aspect of the invention is a computerreadable medium having recorded thereon at least 2, 5, 10, 15, or 20 ofthe sequences as set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Embodiments of the invention include systems (e.g., internet basedsystems), particularly computer systems which store and manipulate thesequence information described herein. One example of a computer system100 is illustrated in block diagram form in FIG. 1. As used herein, “acomputer system” refers to the hardware components, software components,and data storage components used to analyze a nucleotide sequence of anucleic acid sequence as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide sequenceas set forth in the Group B amino acid sequences. The computer system100 typically includes a processor for processing, accessing andmanipulating the sequence data. The processor 105 can be any well-knowntype of central processing unit, such as, for example, the Pentium IIIfrom Intel Corporation, or similar processor from Sun, Motorola, Compaq,AMD or International Business Machines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data, and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular embodiment, the computer system 100 includes aprocessor 105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some embodiments, the computer system100 further includes one or more data retrieving device 118 for readingthe data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet),etc. In some embodiments, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc., containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences, and sequencessubstantially identical thereto, (such as search tools, compare tools,and modeling tools, etc.) may reside in main memory 115 duringexecution.

In some embodiments, the computer system 100 may further comprise asequence comparison algorithm for comparing a nucleic acid sequence asset forth in Group A nucleic acid sequences, and sequences substantiallyidentical thereto, or a polypeptide sequence as set forth in Group Bamino acid sequences, and sequences substantially identical thereto,stored on a computer readable medium to a reference nucleotide orpolypeptide sequence(s) stored on a computer readable medium. A“sequence comparison algorithm” refers to one or more programs which areimplemented (locally or remotely) on the computer system 100 to comparea nucleotide sequence with other nucleotide sequences and/or compoundsstored within a data storage means. For example, the sequence comparisonalgorithm may compare the nucleotide sequences of a nucleic acidsequence as set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin Group B amino acid sequences, and sequences substantially identicalthereto, stored on a computer readable medium to reference sequencesstored on a computer readable medium to identify homologies orstructural motifs. Various sequence comparison programs identifiedelsewhere in this patent specification are particularly contemplated foruse in this aspect of the invention. Protein and/or nucleic acidsequence homologies may be evaluated using any of the variety ofsequence comparison algorithms and programs known in the art. Suchalgorithms and programs include, but are by no means limited to,TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul, et al., J. Mol.Biol., 215(3):403-410, 1990; Thompson, et al., Nucleic Acids Res.,22(2):4673-4680, 1994; Higgins, et al., Methods Enzymol., 266:383-402,1996; Altschul, et al., J. Mol. Biol., 215(3):403-410, 1990; Altschul,et al., Nature Genetics, 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math., 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol, 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA, 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project. Atleast twenty-one other genomes have already been sequenced, including,for example, M. genitalium (Fraser, et al., 1995), M. jannaschii (Bult,et al., 1996), H. influenzae (Fleischmann, et al., 1995), E. coli(Blattner, et al., 1997), and yeast (S. cerevisiae) (Mewes, et al.,1997), and D. melanogaster (Adams, et al., 2000). Significant progresshas also been made in sequencing the genomes of model organism, such asmouse, C. elegans, and Arabidopsis sp. Several databases containinggenomic information annotated with some functional information aremaintained by different organization, and are accessible via theinternet.

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul, et al., Nuc. Acids Res., 25:3389-3402,1977, and Altschul, et al., J. Mol. Biol., 215:403-410, 1990,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915,1989) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA, 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01, and most preferably less than about 0.001.

In one embodiment, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet, etal., Science, 256:1443-1445, 1992; Henikoff and Henikoff, Proteins,17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also beused (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In someembodiments, the parameters may be the default parameters used by thealgorithms in the absence of instructions from the user.

FIG. 2 is a flow diagram illustrating one embodiment of a process 200for comparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence as set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto, or a polypeptide sequence as set forthin Group B amino acid sequences, and sequences substantially identicalthereto, a data storage device having retrievably stored thereonreference nucleotide sequences or polypeptide sequences to be comparedto a nucleic acid sequence as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto, and a sequence comparer forconducting the comparison. The sequence comparer may indicate a homologylevel between the sequences compared or identify structural motifs inthe above described nucleic acid code of Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences, and sequencessubstantially identical thereto, or it may identify structural motifs insequences which are compared to these nucleic acid codes and polypeptidecodes. In some embodiments, the data storage device may have storedthereon the sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or moreof the nucleic acid sequences as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto, or thepolypeptide sequences as set forth in Group B amino acid sequences, andsequences substantially identical thereto.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence as set forth in Group A nucleicacid sequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto, and a reference nucleotidesequence. The method including reading the nucleic acid code or thepolypeptide code and the reference nucleotide or polypeptide sequencethrough the use of a computer program which determines homology levelsand determining homology between the nucleic acid code or polypeptidecode and the reference nucleotide or polypeptide sequence with thecomputer program. The computer program may be any of a number ofcomputer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences as set forth in theGroup A nucleic acid sequences, or the polypeptide sequences as setforth in the Group B amino acid sequences through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 ina computer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of Group A nucleic acidsequences, and sequences substantially identical thereto, differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or a nucleic acid sequence as set forth inGroup A nucleic acid sequences, and sequences substantially identicalthereto. In one embodiment, the computer program may be a program whichdetermines whether a nucleic acid sequence as set forth in Group Anucleic acid sequences, and sequences substantially identical thereto,contains a single nucleotide polymorphism (SNP) with respect to areference nucleotide sequence.

Accordingly, another aspect of the invention is a method for determiningwhether a nucleic acid sequence as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto, differs at oneor more nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, or 40 or more of the nucleic acidsequences as set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto, and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other embodiments the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence asset forth in the Group A nucleic acid sequences or a polypeptidesequence as set forth in Group B amino acid sequences, and sequencessubstantially identical thereto.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence as set forth in Group A nucleicacid sequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto. In one embodiment, theidentifier may comprise a program which identifies an open reading framein a nucleic acid sequence as set forth in Group A nucleic acidsequences, and sequences substantially identical thereto.

FIG. 5 is a flow diagram illustrating one embodiment of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence.

It should be noted, that if the feature attribute is not found in thefirst sequence at the decision state 316, the process 300 moves directlyto the decision state 320 in order to determine if any more featuresexist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence as set forth in Group A nucleicacid sequences, and sequences substantially identical thereto, or apolypeptide sequence as set forth in Group B amino acid sequences, andsequences substantially identical thereto, comprising reading thenucleic acid code(s) or polypeptide code(s) through the use of acomputer program which identifies features therein and identifyingfeatures within the nucleic acid code(s) with the computer program. Inone embodiment, computer program comprises a computer program whichidentifies open reading frames. The method may be performed by reading asingle sequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of thenucleic acid sequences as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or the polypeptidesequences as set forth in Group B amino acid sequences, and sequencessubstantially identical thereto, through the use of the computer programand identifying features within the nucleic acid codes or polypeptidecodes with the computer program.

A nucleic acid sequence as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences, and sequencessubstantially identical thereto, may be stored and manipulated in avariety of data processor programs in a variety of formats. For example,a nucleic acid sequence as set forth in Group A nucleic acid sequences,and sequences substantially identical thereto, or a polypeptide sequenceas set forth in Group B amino acid sequences, and sequencessubstantially identical thereto, may be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2, SYBASE, or ORACLE. In addition, many computer programsand databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence as set forth inGroup A nucleic acid sequences, and sequences substantially identicalthereto, or a polypeptide sequence as set forth in Group B amino acidsequences, and sequences substantially identical thereto. The followinglist is intended not to limit the invention but to provide guidance toprograms and databases which are useful with the nucleic acid sequencesas set forth in Group A nucleic acid sequences, and sequencessubstantially identical thereto, or the polypeptide sequences as setforth in Group B amino acid sequences, and sequences substantiallyidentical thereto.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul, et al., J. Mol. Biol., 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag, et al., Comp. App. Biosci., 6:237-245,1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DB Access (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwent'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase, and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups, and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivitization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies, and compounds can besynthesized and tested free in solution using virtually any type ofscreening assay. It is important to note, that the high degree ofspecificity of enzyme reactions on functional groups allows for the“tracking” of specific enzymatic reactions that make up thebiocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular embodiment, the invention provides a method formodifying small molecules, comprising contacting a polypeptide encodedby a polynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library, and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity isoptionally repeated. The biocatalytic reactions are conducted with agroup of biocatalysts that react with distinct structural moieties foundwithin the structure of a small molecule, each biocatalyst is specificfor one structural moiety or a group of related structural moieties; andeach biocatalyst reacts with many different small molecules whichcontain the distinct structural moiety.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Site-Saturation Mutagenesis

To accomplish site-saturation mutagenesis every residue (317) of adehalogenase enzyme (SEQ ID NO:2) encoded by SEQ ID NO:1 was convertedinto all 20 amino acids by site directed mutagenesis using 32-folddegenerate oligonucleotide primers, as follows:

-   -   1. A culture of the dehalogenase expression construct was grown        and a preparation of the plasmid was made.    -   2. Primers were made to randomize each codon—they have the        common structure X₂₀NN(G/T)X₂₀, wherein X₂₀ represents the 20        nucleotides of the nucleic acid sequence of SEQ ID NO:1 flanking        the codon to by changed.    -   3. A reaction mix of 25 Tl was prepared containing ˜50 ng of        plasmid template, 125 ng of each primer, 1× native Pfu buffer,        200 TM each dNTP and 2.5 U native Pfu DNA polymerase.    -   4. The reaction was cycled in a Robo96 Gradient Cycler as        follows:        -   Initial denaturation at 95° C. for 1 min;        -   20 cycles of 95° C. for 45 sec, 53° C. for 1 min and 72° C.            for 11 min; and        -   Final elongation step of 72° C. for 10 min    -   5. The reaction mix was digested with 10 U of DpnI at 37° C. for        1 hour to digest the methylated template DNA.    -   6. Two Tl of the reaction mix were used to transform 50 Tl of        XL1-Blue MRF′ cells and the entire transformation mix was plated        on a large LB-Amp-Met plate yielding 200-1000 colonies.    -   7. Individual colonies were toothpicked into the wells of        384-well microtiter plates containing LB-Amp-IPTG and grown        overnight.    -   8. The clones on these plates were assayed the following day.

Example 2 Dehalogenase Thermal Stability

This invention provides that a desirable property to be generated bydirected evolution is exemplified in a limiting fashion by an improvedresidual activity (e.g., an enzymatic activity, an immunoreactivity, anantibiotic activity, etc.) of a molecule upon subjection to alteredenvironment, including what may be considered a harsh environment, for aspecified time. Such a harsh environment may comprise any combination ofthe following (iteratively or not, and in any order or permutation): anelevated temperature (including a temperature that may causedenaturation of a working enzyme), a decreased temperature, an elevatedsalinity, a decreased salinity, an elevated pH, a decreased pH, anelevated pressure, a decreased pressure, and an change in exposure to aradiation source (including uv radiation, visible light, as well as theentire electromagnetic spectrum).

The following example shows an application of directed evolution toevolve the ability of an enzyme to regain or retain activity uponexposure to an elevated temperature.

Every residue (317) of a dehalogenase enzyme was converted into all 20amino acids by site directed mutagenesis using 32-fold degenerateoligonucleotide primers, as described above. The screening procedure wasas follows:

-   -   1. Overnight cultures in 384-well plates were centrifuged and        the media removed. To each well was added 0.06 mL 1 mM Tris/SO₄        ²⁻ pH 7.8.    -   2. A robot made 2 assay plates from each parent growth plate        consisting of 0.02 mL cell suspension.    -   3. One assay plate was placed at room temperature and the other        at elevated temperature (initial screen used 55° C.) for a        period of time (initially 30 minutes).    -   4. After the prescribed time 0.08 mL room temperature substrate        (TCP saturated 1 mM Tris/SO₄ ²⁻ pH 7.8 with 1.5 mM NaN₃ and 0.1        mM bromothymol blue) was added to each well.        TCP=trichloropropane.    -   5. Measurements at 620 nm were taken at various time points to        generate a progress curve for each well.    -   6. Data were analyzed and the kinetics of the cells heated to        those not heated were compared. Each plate contained 1-2 columns        (24 wells) of un-mutated 20F12 controls.    -   7. Wells that appeared to have improved stability were regrown        and tested under the same conditions.

Following this procedure clones having mutations that conferredincreased thermal stability on the enzyme were sequenced to determinethe exact amino acid changes at each position that were specificallyresponsible for the improvement. Mutants having a nucleic acid sequenceas set forth in SEQ ID NO's: 5 and 7 and polypeptide sequences as setforth in SEQ ID NO's: 6 and 8, respectively, were identified. Thethermal mutant at position G182V (SEQ ID NO: 6) can also be a glutamate(Q) with similar increased thermal stability. Similarly, the P302Amutation could be changed to leucine (L), serine (S), lysine (K) orarginine (R). These variants (as well as those below) are encompassed bythe present invention.

Following this procedure nine single site mutations appeared to conferincreased thermal stability. Sequence analysis showed that the followingchanges were beneficial: D89G; F91S; T159L; G182Q, G182V; 1220L; N238T;W251Y; P302A, P302L, P302S, P302K; P302R/S306R. Only two sites (182 and302) had more than one substitution. The first 5 on the list werecombined (using G182Q) into a single gene.

Thermal stability was assessed by incubating the enzyme at the elevatedtemperature (55° C. and 80° C.) for some period of time and activityassay at 30° C. Initial rates were plotted vs. time at the highertemperature. The enzyme was in 50 mM Tris/SO₄ pH 7.8 for both theincubation and the assay. Product (Cl⁻) was detected by a standardmethod using Fe(NO₃)₃ and HgSCN. The dehalogenase of SEQ ID NO: 2 wasused as the de facto wild type. The apparent half-life (T_(1/2)) wascalculated by fitting the data to an exponential decay function.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

What is claimed is:
 1. An isolated, synthetic or recombinant nucleicacid comprising (a) a nucleic acid sequence having at least 90% sequenceidentity to SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 43, 45, and 47, wherein the nucleic acid encodesat least one polypeptide having a dehalogenase activity or encodes atleast one polypeptide capable of generating an antibody response byadministering the polypeptide to an animal; or (b) a sequencecomplementary to (a).
 2. An isolated, synthetic or recombinantpolypeptide having a dehalogenase activity comprising an amino acidsequence having at least 90% sequence identity to SEQ ID NOS: 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 43, 45,and 47.